Identification, molecular cloning, and characterization of a novel GABAA receptor-associated protein, GRIF-1
Identification, molecular cloning, and characterization of a novel GABAA receptor-associated protein, GRIF-1
复制标题
DOI：
10.1074/jbc.m200438200
复制
发表时间：
2002-08-16
影响因子：
4.8
通讯作者：
Stephenson, FA
中科院分区：
文献类型：
--
作者：
Beck, M;Brickley, K;Stephenson, FA
一个新的913个氨基酸的蛋白，γ-氨基丁酸A型（GABA（A））受体相互作用因子-1（GRIF-1），已被克隆和鉴定为GABA（A）受体相关蛋白凭借其特异性相互作用的GABA（A）受体β 2亚基的细胞内环在酵母双杂交试验。GRIF-1与已知功能的蛋白质没有同源性，但它是人ALS 2CR 3/KIAA 0549基因的大鼠直向同源物。GRIF-1表达为两种选择性剪接形式，GRIF-1a和C末端截短形式，GRIF-1b。GRIF-1 mRNA具有广泛的分布，主要转录物大小为6.2 kb。GRIF-1a蛋白仅在可兴奋的组织中表达，即脑、心脏和骨骼肌M-r的主要免疫反应性条带类似于115和106 kDa，并且仅在肌肉和心脏中表达另外的88-kDa种类。当在人胚肾293细胞中表达时，GRIF-1a产生三条免疫反应性条带，M-r类似于115、106和98 kDa。GRIF-1a和α 1 β 2 γ 2 GABA（A）受体在哺乳动物细胞中的共表达揭示了细胞质中的一些共定位。抗FLAG-琼脂糖特异性沉淀来自与GRIF-1a（FLAG）和β 2亚基克隆共转染的人胚肾293细胞的GRIF-1（FLAG）和GABAA受体β 2亚基。此外，固定化GRIF-1-（8-633）在体外特异性沉淀成年大鼠脑洗涤剂提取物中的GABA（A）受体α 1和β 2亚基免疫反应性。使用酵母双杂交报告基因测定对相应的GABA（A）受体β 2亚基/GRIF-1结合结构域进行作图。GRIF-1作为GABA（A）受体β 2亚单位运输因子的可能作用被提出。
A novel 913-amino acid protein, gamma-aminobutyric acid type A (GABA(A)) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA(A) receptor-associated protein by virtue of its specific interaction with the GABA(A) receptor beta2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M-r similar to 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M-r similar to 115, 106, and 98 kDa. Co-expression of GRIF-1a and alpha1beta2gamma2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABAA receptor beta2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta2 subunit clones. Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha1 and beta2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA(A) receptor beta2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA(A) receptor beta2 subunit trafficking factor is proposed.